Actinium-225 targeted alpha particle therapy for prostate cancer

Targeted alpha particle therapy (TAT) has emerged as a promising strategy for the treatment of prostate cancer (PCa). Actinium-225 (225Ac), a potent alpha-emitting radionuclide, may be incorporated into targeting vectors, causing robust and in some cases sustained antitumor responses. The development of radiolabeling techniques involving EDTA, DOTA, DOTPA, and Macropa chelators has laid the groundwork for advancements in this field. At the forefront of clinical trials with 225Ac in PCa are PSMA-targeted TAT agents, notably [225Ac]Ac-PSMA-617, [225Ac]Ac-PSMA-I&T and [225Ac]Ac-J591. Ongoing investigations spotlight [225Ac]Ac-hu11B6, [225Ac]Ac-YS5, and [225Ac]Ac-SibuDAB, targeting hK2, CD46, and PSMA, respectively. Despite these efforts, hurdles in 225Ac production, daughter redistribution, and a lack of suitable imaging techniques hinder the development of TAT. To address these challenges and additional advantages, researchers are exploring alpha-emitting isotopes including 227Th, 223Ra, 211At, 213Bi, 212Pb or 149Tb, providing viable alternatives for TAT.


Introduction
Prostate cancer (PCa) is the most common non-cutaneous malignancy among men, and a significant threat to public health.PCa is projected to be diagnosed in approximately 299,010 men within the United States in 2024, accounting for approximately 29% of all malignancies [1].The lethality of prostate cancer stems from its capacity to metastasize, leading to significant health challenges and loss of life.Despite the advances in diagnostic techniques, including prostate-specific antigen (PSA) screening, a substantial proportion of patients still present with aggressive forms of the disease, necessitating better and alternative therapeutic strategies.Effective management of prostate cancer involves a diverse array of treatment modalities tailored to specific characteristics and disease stage [2].For localized or early-stage prostate cancer, treatment options may include active surveillance, cryosurgery, brachytherapy or external beam radiotherapy, radical prostatectomy (surgical removal of the prostate gland), hormone therapy, or other focal therapies aimed at targeting specific areas of the prostate [2,3].However, in metastatic disease, curative options become limited, and the challenges of treatment increase.Conventional therapies, such as surgery, radiation, and hormone therapy, may effectively control localized disease or delay progression, but they often are not cancer-specific and come with significant side effects, which may not be suitable for all patients.
The urgency for more precise and effective treatment options has driven the emergence of targeted therapies in oncology.These therapies aim to exploit specific molecular features of cancer cells to deliver highly potent and selective treatments.Prostate-Specific Membrane Antigen (PSMA), a type

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II transmembrane glycoprotein has revolutionized diagnostic, prognostic, and therapeutic approaches [4].With its elevated and selective overexpression in prostate cancer cells, PSMA stands as an important target for precision medicine strategies.The ectodomain of PSMA exhibits glutamate carboxypeptidase enzymatic activity, mediating the cleavage of N-acetylated-L-aspartyl-L-glutamate (NAAG), thereby potentially influencing tumor microenvironment modulation [5].Its overexpression is attributed to androgen receptor signaling and other molecular mechanisms and substantiates its potential as a therapeutic target.PSMA-targeted imaging using radiopharmaceuticals has revolutionized prostate cancer detection [6].PSMA targeted PET imaging is now standard of care in prostate cancer.
More recently, PSMA targeted radiopharmaceutical therapy has emerged as a powerful therapeutic modality in advanced metastatic castration resistant prostate cancer.The phase II TheraP study compared [ 177 Lu]Lu-PSMA-617 with cabazitaxel in metastatic castration-resistant prostate cancer [7].[ 177 Lu]Lu-PSMA-617 showed higher PSA responses (66% vs 37%) and fewer severe adverse events (33% vs 53%) compared to cabazitaxel [8].The findings suggest [ 177 Lu]Lu-PSMA-617 as a promising and well-tolerated alternative to cabazitaxel for this patient population.Subsequently, the VISION trial marked a substantial advancement in the ongoing battle against mCRPC.This pivotal international effort was a randomized, open-label phase III trial that successfully recruited 831 men diagnosed with PSMA-positive mCRPC [9].The participants were divided into two treatment groups: one receiving [ 177 Lu]Lu-PSMA-617 in conjunction with the standard of care (n=551), and the other undergoing the standard of care alone (n=280).The groundbreaking results of the trial revealed a significant improvement in progression-free and overall survival for those treated with [ 177 Lu]Lu-PSMA-617 alongside standard of care compared to those receiving standard of care alone.
Figure 1 depicts the most common components of the various radioligand modalities including small molecules, peptides, and antibody-based agents for radiotheranostics.Positron Emission Tomography (PET) radiotracers, such as [ 68 Ga]Ga-PSMA-11 and [ 18 F]-DCFPyL, exhibit high specificity and sensitivity in identifying metastatic lesions and guiding treatment decisions, thereby circumventing the limitations of conventional imaging techniques [10].Recently, the FDA has approved [ 177 Lu]Lu-PSMA-617 (Pluvicto) for patients with advanced metastatic castration-resistant prostate cancer (mCRPC) previously treated with chemotherapy and antiandrogen therapy [11].However, the TheraP study, as well as the phase III VISION study, further indicated that 17% and 30% of patients had inherent resistance to the [ 177 Lu]Lu-PSMA-617, respectively [12].
Targeted alpha therapy (TAT) harnesses the unique properties of alpha particle radiation for cancer treatment.Alpha particles are high-energy (LET= 80 keV/µm), highly charged particles that possess a short range (40-100 µm), making them ideal for localized and precise targeting of cancer cells (Figure 2) [13].Unlike beta particles (0.2 keV/µm, 0.05-12 mm), alpha particles deposit a significant amount of energy over a very short distance, resulting in potent and localized damage to tumor cells.This characteristic allows for targeted delivery and reduced cross-fire effect to surrounding healthy tissues.
Actinium-225 ( 225 Ac) is a promising alphaemitting radionuclide for prostate cancer targeted radiotherapy. 225Ac has a half-life of 9.92 days, during which it decays through a series of short-lived alpha and beta-emitting daughters (Figure 3).This decay chain results in the release of four net alpha particles per decay event, maximizing the therapeutic potential of 225 Ac.In this review paper, we aim to provide a comprehensive understanding of Actinium-225-based targeted alpha therapy for prostate cancer treatment.By exploring the principles of alpha particle radiation and its advantages in cancer therapy, we seek to emphasize the significance of targeted therapies in prostate cancer treatment.Furthermore, we will delve into the properties of 225 Ac, the radiolabeling techniques utilized for its efficient delivery, and its role in preclinical and clinical studies.Additionally, we will evaluate the efficacy, safety considerations, and challenges associated with 225 Ac-based TAT for prostate cancer.
The approval of 223 RaCl2 (Xofigo) by the FDA in 2013 for managing prostate cancer bone metastases in castration-resistant prostate cancer patients sparked a resurgence of interest in TATs.Despite the successful use of 223 RaCl2, incorporation of 223 Ra into targeted agents was not established due to its challenging chelation chemistry [20].However, other α-emitting radionuclides, including 227 Th, 225 Ac, 213 Bi, 212 Bi, 212 Pb, and 211 At, have more suitable chemical properties for attachment to targeting vectors.The use of these radioisotopes requires the formation of a thermodynamically stable and kinetically inert complex with a chelator.Limited knowledge of the Ac 3+ ion coordination chemistry made it difficult to find optimum chelators for a stable complex.Additionally, with a large ionic radius (112 pm, 1.12 Å) and low charge-to-ionic ratio, Ac 3+ tends to display weak electrostatic interactions with the chelator [20].The daughter isotopes from 225 Ac also experience recoil energy due to the conservation of momentum, which leads to a break in the coordination and subsequent release of the daughter isotope from the chelator [21].Therefore, finding a chelator providing sufficient stability has been an active challenge in the field of TAT with 225 Ac.Additionally, chelator development research was slowed due to limited access to a regular supply of 225 Ac.
Since the early 1990s, extraction of 225 Ac from 229 Th has assumed a pivotal role in the production of both 225 Ac and 213 Bi.Subsequently, clinical and preclinical studies have depended on the 225 Ac derived through the decay of 229 Th [19].Globally, there exist three sources of 229 Th facilitating the generation of 225 Ac of clinical relevance.These sources are strategically located at the Directorate for Nuclear Safety and Security of the JRC of the European Commission in Karlsruhe, Germany, the Oak Ridge National Laboratory (ORNL) in the United States, and the Institute of Physics and Power Engineering (IPPE) in Obninsk, Russia.The origins of the 229 Th sources trace back to aged 233 U, initially synthesized for weaponry applications via neutron irradiation of natural 232 Th.
The escalating demand for 225 Ac has drawn the attention of researchers, medical practitioners, and the global patient population, all awaiting the provision of this crucial radionuclide.Their focus revolves around its subsequent application in tailoring patient-specific therapeutic regimens through radiolabeling methodologies.There has been a global response to produce 225 Ac to meet the potential future demands fueled by TAT.A comprehensive update on 225 Ac production is provided later in this review.

Actinium-225 based TAT Agents for Prostate Cancer Tumor Models
Preclinical studies evaluating small molecule TAT agents PSMA is a prime target for radioligand therapy (RLT).Radionuclides like 177 Lu and 225 Ac, conjugated with PSMA-targeting ligands, enable selective irradiation of tumor cells while minimizing damage to healthy tissues.Since the alpha particle possesses high LET and shorter path length, TAT with 225 Ac may demonstrate enhanced therapeutic efficacy.A range of α-emitting radionuclides, namely 149 Tb, 211 At, 212 Pb/ 212 Bi, 225 Ac, and 227 Th, are currently undergoing rigorous assessment for PSMA-targeted alpha particle therapy.PSMA is also a unique target in that it accommodates bulky groups conjugated to the molecule that engages the protein binding site.This property has been accessed to develop a variety imaging and therapeutic PSMA targeted agents.
The utilization of mouse models in PCa research has yielded valuable insights into cancer mechanisms, therapeutic strategies, and treatment advancements.However, the choice of model often limits its clinical applicability.Subcutaneous xenograft models facilitate convenient tracking of tumors and responses but cannot mimic metastatic progression.On the other hand, orthotopic and systemic models offer a clinically relevant context despite being more challenging to establish and monitor.In the context of PSMA-targeted alpha therapy within a murine metastatic prostate cancer model, the timing of treatment emerged as a pivotal factor influencing therapeutic outcomes [28].Stuparu et al. showed that administering [ 225 Ac]Ac-PSMA-617 in the early stage of C4-2 metastatic tumors holds promise for treating disease with significant survival benefit [28].The use of systemic models is preferable due to their ability to replicate clinical conditions better and provide a precise assessment of treatment responses and interactions.
Due to its relatively low molecular weight, PSMA is readily eliminated through the renal excretion pathway.While a high clearance rate is advantageous for imaging radiotracers, it may inadvertently lead to diminished radiation dose deposition within tumors.To enhance the radiation dose targeted at tumors, modifications are being explored in PSMA ligands to prolong their circulation and tumor retention times.A study by Meyer et al. demonstrated that incorporating an extended linker with supplementary naphthyl groups enhances ligand binding to albumin, thereby extending circulating half-life [29].Using a similar strategy, Busslinger et al. developed ibuprofen containing ligand [ 225 Ac]Ac-SibuDAB, which showed similar radiolabeling and hematological effect as [ 225 Ac]Ac-PSMA-617, whereas the tumor uptake of the [ 225 Ac]Ac-SibuDAB doubled at 48 h as compared to [ 225 Ac]Ac-PSMA-617 (64 ± 11% IA/g vs. 31 ± 3% IA/g), resulting in improved therapeutic response and overall survival in animal models [30].Another study with macropa chelator conjugated to albumin binding unit (4-(p-iodophenyl)butyrate) and one or two PSMA ligands also showed that the uptake of an albumin-bound [ 225 Ac]Ac-PSMA molecule was four times higher than the non-albumin binding counterpart [31].A similar albumin-binding PSMA-targeting ligand, RPS-074, labeled with 225 Ac, achieved complete remission in 6 out of 7 LNCaP xenografts with a single administration of 148 kBq [32].

Antibody-based Radioimmunotherapy agents for Preclinical Tumors of PCa
Radioimmunotherapy (RIT) combines the precision of monoclonal antibodies with the robust cell-destructive potential of radiation therapy.Large molecules like monoclonal antibodies with higher molecular weights typically exhibit extended circulation within the bloodstream and prolonged bodily retention.By incorporating 225 Ac, characterized by its long half-life, onto RIT agent, the therapy gains the added advantage of sustained radiation emission over an extended timeframe.The selection of the chelator for radioimmunotherapy becomes essential, as it plays a crucial role in securely binding and holding the radioactive isotope to the monoclonal antibody.Seminal work by McDevitt et al. demonstrated that the internalizing anti-PSMA antibody [ 225 Ac]Ac-J591 acts as a nanogenerator, producing a series of α particles for maximum radiation dose [33].In vitro experiments conducted in this study revealed that internally localized [ 225 Ac]Ac-J591 in LNCaP cells retained the daughter isotopes along with their corresponding α emissions.However, it was noted that daughters produced by [ 225 Ac]Ac-J591 bound to the cell surface were likely to be transferred to other sites.Additionally, [ 225 Ac]Ac-J591 exhibited a significant anti-tumor response in LNCaP tumor models, leading to improved survival rates and a substantial decrease in PSA levels.This agent is now in clinical trials as outlined below.
The significance of tumor PSMA expression is underscored by its far-reaching implications for both diagnostic and therapeutic strategies.The absence or downregulation of PSMA directly impacts the efficacy of PSMA-targeted approaches [34].Consequently, a pressing need has arisen to explore alternative targets that can facilitate the development of efficacious theranostic strategies.In response to this, notable progress has been achieved through the development of antibodies that target prostate cancer regardless of their PSMA status.One instance of this approach is the development of a full-length IgG antibody targeting CD46, as demonstrated by Su et al [35].CD46 protein was found to be overexpressed on PCa tissues, and the anti-CD46 antibody effectively binds to PCa irrespective of PSMA expression [35].For TAT of prostate cancer, the CD46 targeting [ 225 Ac]Ac-DOTA-YS5 antibody, labeled with 225 Ac, was harnessed [36].The therapeutic agent proved efficacious in reducing tumor volume, exhibiting efficacy against both cell-derived (22Rv1 and DU145) and patient-derived adenocarcinoma (LTL-484) and neuroendocrine prostate cancer (LTL-545) models.Bidkar et al. also showed that fractionation of [ 225 Ac]Ac-DOTA-YS5 into multiple smaller doses of 0.125 µCi results in a better therapeutic response with lesser toxicity.In addition to this, a surrogate imaging probe [ 89 Zr]Zr-DFO-YS5 has been developed to image CD46-positive tumors [37].Recently, Bobba et al. utilized a PEGylated Macropa chelator (18-membered macrocyclic) conjugated to an anti-CD46 antibody, demonstrating highly efficient radiolabeling (96% yield) at room temperature within a short timeframe of 5 minutes [38].This approach surpassed the performance of DOTA conjugates and exhibited superior antitumor efficacy.The CD46 target has proven effective for the tumor-targeted delivery of radiation.In a related study, Li et al. demonstrated the therapeutic efficacy of a similar alpha emitting isotope, 212 Pb, conjugated to an anti-CD46 antibody ([ 212 Pb]Pb-TCMC-YS5) [39].This novel construct showed promising therapeutic effects with a single dose in both subcutaneous mCRPC cell line-derived and patient-derived xenograft models.
The hK2 is a serine protease expressed in prostate tissue that has an 80% amino acid sequence similar to PSA.The antibody hu11B6 targets hK2, an epitope expressed exclusively on prostate tissue and cancer in vivo.Additionally, it is regulated by the androgen receptor (AR) activity.McDevitt et al. prepared the [ 225 Ac]Ac-hu11B6 (with DOTA chelator), which delivers radiation specifically to cancer cells, thereby initiating a DNA damage response (DDR) [40].Subsequently, the DDR causes upregulation of the hK2, resulting in feed-forward alpha particle radiotherapy.The [ 225 Ac]Ac-hu11B6 therapy took advantage of the unique phenomenon of increasing expression of AR and hK2 from alpha particleinduced DNA damage in the LNCaP-AR tumor model.The hK2 targeting antibody has also been utilized to make an imaging agent [ 89 Zr]Zr-hu11B6 to use as a surrogate reporter for [ 225 Ac]Ac-hu11B6 and beta particle therapy agent [ 177 Lu]Lu-m11B6 [40,41].The authors also studied the expression of AR-governed biomarkers in treatment-naïve tumors vs. relapsing tumors.Genes under the control of androgen receptor (AR), namely FOLH1, KLK3, PMEPA1, and SPOCK1, displayed a down-regulated pattern, whereas a subset of androgen-repressed genes, including PMP22, CAMK2N1, and UGT2B17, demonstrated an up-regulated expression [42].Unlike the loss of PSMA in treatment-emergent neuroendocrine/small cell prostate cancer, hK2 (KLK2) expression was intact in the tumors of mice from [ 225 Ac]Ac-hu11B6 treatment.The hK2 targeting alpha particle RIT agent ([ 225 Ac]Ac-DOTA-h11B6) is currently under phase I clinical trial [43].
Since the expression of hK2 and PSA is regulated due to AR activity, it has led to the utilization of PSA as a target for RIT using an antibody.Veach et al. compared the therapeutic efficiency of two types of antibodies targeting PSA: high linear energy transfer (LET) [ 225 Ac]Ac-hu5A10 with an LET of approximately 100 keV/µm, and low LET [ 90 Y]Y-hu5A10 with a LET of about 0.2 keV/µm [44].This comparison conducted in LNCaP-AR xenografts showed that both the high LET and low LET antibodies resulted in improved median survival when compared to saline control (32 days).Notably, the high LET antibody achieved a significantly higher median survival (188 days) compared to the low LET antibody, showcasing the superiority of [ 225 Ac]Ac-hu5A10.Moreover, the study found that a substantial percentage of animals exhibited a complete response to the high LET [ 225 Ac]Ac-hu5A10 (38.9%), whereas the low LET [ 90 Y]Y-hu5A10 achieved a complete response in only 11.1% of animals.
In the context of addressing solid tumors, an additional emerging therapeutic strategy involves inhibiting tumor angiogenesis.An intriguing approach revolves around vascular endothelial (VE) cadherin, a molecule exclusive to vascular endothelial cells that are consistently expressed throughout the vascular network.VE plays a pivotal role in the establishment of adherens junctions between neighboring endothelial cells.A monoclonal antibody, E4G10, has been developed to specifically bind to an epitope exposed solely to the monomeric, unengaged form of VE-cadherin [45].This epitope becomes concealed upon transdimerization, leading to the formation of inter-cellular junctions.This distinctive feature permits the precise targeting of endothelial cells within nascent tumor vasculature, presenting a selective strategy to hinder tumor growth.Using this approach, Jaggi et al. designed [ 225 Ac]Ac-E4G10 antibody targeting neovasculature, which showed a significant reduction of LNCaP subcutaneous tumor xenografts.
While RIT agents effectively target tumors, a notable concern is the prolonged circulation time, particularly in the context of 225 Ac delivery.Optimizing the circulation duration is crucial, and in certain cases, a faster clearance would be beneficial for improved therapeutic outcomes.

Clinical Studies Testing 225 Ac TAT Agents in Prostate Cancer Patients [ 225 Ac]Ac-PSMA-617
In a first description of the use of [ 225 Ac]Ac-PSMA-617 for patients, Kratochwil et al. presented two cases of mCRPC patients who had previously received either conventional chemotherapy or [ 177 Lu]Lu-PSMA-617 treatment [46].Notably, the first patient exhibited the disappearance of PSMA-positive lesions on PSMA PET/CT and a remarkable decline in PSA levels from over 3,000 ng/mL to 0.26 ng/mL within two months of treatment of [ 225 Ac]Ac-PSMA-617.Subsequent consolidation therapy with [ 225 Ac]Ac-PSMA-617 led to further PSA reduction and a complete PSMA PET/CT imaging response.The second patient, having exhausted conventional treatments and [ 177 Lu]Lu-PSMA-617 regimens, entered the study with elevated PSA levels at 419 ng/mL.Treatment with [ 225 Ac]Ac-PSMA-617 across three cycles led to a full response in this patient, with PSA levels reaching below 0.1 ng/mL.These findings underscored the potential of [ 225   [50].Thus, a decrease of PSA in the first few cycles could be followed by a de-escalation of [ 225 Ac]Ac-PSMA-617 dose to manage the xerostomia, simultaneously inhibiting tumor growth.The small molecule PSMA ligands show uptake in salivary glands leading to radiation-induced xerostomia.Various strategies are being employed to reduce the uptake of salivary gland hypofunction after RLT or other radiation therapies.Among these options are the external cooling of the glands [51] and the potential use of botulinum toxin A injections to reduce PSMA-targeting compound uptake by lowering salivary gland metabolism [52].Another option involves investigating polyglutamate as a PSMA-binding competitor, with promising initial clinical outcomes [53].Restoring salivary gland function is a third approach, with ongoing investigations into gene transfer of the aquaporin-1 gene [54].Sour stimulation and sialendoscopy with dilatation, saline irrigation, and steroid injections have also been explored, demonstrating beneficial effects on radiation-induced inflammation and quality of life [55].However, even with sialendoscopic support following multiple cycles of TAT, salivary gland function remained diminished, and xerostomia persisted.In addition to this, a combination of low-activity [ 225 Ac]Ac-PSMA-617 with full-activity doses [ 177 Lu]Lu-PSMA-617 shows improved efficacy and good tolerability in heavily pre-treated mCRPC patients.The approach appeared to enhance treatment response and reduce the xerostomia severity, suggesting that 'tandem therapy' could be an effective strategy [56].Furthermore, the replacement of Glu from the PSMA ligand (Lys-urea-Glu) with Asp or Aad (L-2-aminoadipic acid) has led to the development of a new class of radiotracer probe or therapy agent aimed at reducing salivary gland or kidney uptake.Using this strategy, Kuo et al. designed the albumin-binding PSMA targeting agent [ 177 Lu]Lu-HTK03149 (Glu replaced with Aad), resulting in significantly higher tumor uptake (145% higher absorbed dose) than [ 177 Lu]Lu-PSMA-617, along with lower uptake in salivary glands (0.23 vs 1.78 %IA/g) and kidneys (79.7 vs 7.67%IA/g) at 1h post-injection [57].
Presently, [ 225 Ac]Ac-PSMA-617 treatment is often being used as a final resort for mCRPC, after extensive pretreatment involving ADT, chemotherapy, and radioligand therapy with 177 Lu-PSMA-617.This heavy pretreatment contributes to tumor resistance while also rendering patients more susceptible to adverse effects and toxicities.Chemotherapy-naïve patients with advanced metastatic prostate carcinoma respond to [ 225 Ac]Ac-PSMA-617 effectively.The treatment-naïve patients displayed a decline of over 90% in their serum PSA levels after undergoing three or four treatment cycles [58].Conversely, patients whose disease had progressed after receiving two or more previous treatments experienced a less favorable decline in PSA levels following the [ 225 Ac]Ac-PSMA-617 treatment.The de-escalation from a fixed activity dose of 8 MBq for the first cycle to 7 MBq to 4 MBq was achieved for the next cycles with positive responses and reduced xerostomia.Using this regimen, a >50% PSA decrease was seen in 91% of patients [50].Molecular changes in the tumor cells including the mutations in the DNA damage repair genes, are considered the factors associated with the resistance to TAT [59].A recent multicenter study conducted by Sathekge et al. investigated the treatment outcomes and adverse effects of [ 225 Ac]Ac-PSMA-617 in mCRPC patients who had undergone with one or more previous lines of treatment [60].The study revealed that 70% of patients showed any decline in PSA, with 57% of patients showing a decline of 50% or more.The most common side effect observed was xerostomia, followed by bone marrow toxicity [60].
Owing to its longer path, beta particles cross the tumor-affected area and affect the surrounding healthy cells (sometimes termed the crossfire effect), potentially resulting in toxicity.[ 177 Lu]Lu-PSMA-617 delivery to bone metastasis may result in significant toxicity to bone marrow leading to hematological complexities.Conversely, alpha particles from [ 225 Ac]Ac-PSMA-617 with a shorter range and high LET are considered more useful for micro-metastatic tumors [61].Therefore, [ 225 Ac]Ac-PSMA-617 has been an effective treatment for brain metastasis of mCRPC [62].

J951 for PSMA Targeted Theranostics
In addition to the developments of the small molecule PSMA targeting ligands, various antibodies targeting PSMA have been studied, which mitigate salivary gland toxicity [63][64][65].Among these, 7E11, a PSMA intracellular domain binding, and J951, an extracellular domain binding antibody, have been extensively tested for clinical applications (Table 2) [63].Since then, [ 89 Zr]Zr-huJ591 has been shown to localize and detect metastatic lesions in prostate cancer patients, while the β or α particle emitter-loaded antibodies are also being tested clinically [66].A single dose or fractionated dose of [ 177 Lu]Lu-J591 showed efficacy in mCRPC patients, with a decline in PSA levels [67,68].Dose escalation studies (NCT04576871, NCT03276572, and NCT0450 6567) are being performed for single and fractionated doses of [ 225 Ac]Ac-J591 in mCRPC patients [69,70].The phase I dose escalation of the [ 225 Ac]Ac-J591 involved 32 patients, dosed at 13.3 to 93.3 KBq/kg [71].The results from this study demonstrated that one patient showed dose-limiting toxicity (grade ≥3 nonhematologic or grade 4 hematological) with 80 KBq/kg dose and hematologic adverse events (grade 3) were in correlation with administered radioactivity.This trial demonstrated safety in administering a single dose of [ 225 Ac]Ac-J591 to patients with pretreated progressive mCRPC.The safety profile, dosimetry, and therapeutic potential of the combination of the small molecule [ 177 Lu]Lu-PSMA I&T and the [ 225 Ac]Ac-J591 are also being studied to utilize the complimentary benefits of two therapies in mCRPC patients [72].

TAT in the hormone-sensitive PCa setting
Various factors such as significant tumor load, low hemoglobin levels, reduced performance condition, and a history of several past therapies all influence RLT outcome and tolerance [77].Consequently, it is hypothesized that PSMA-RLT may be advantageous for patients in an earlier disease stage.A recent prospective phase I study demonstrated promising results, where ten patients in the early stages of hormone-sensitive prostate cancer underwent two cycles of [ 177 Lu]Lu-PSMA-617 RLT [78,79].Ongoing Phase 3 trials, including PSMAfore (NCT04689828), SPLASH (NCT04647526), and ECLIPSE (NCT05204927), are actively evaluating the benefits of [ 177 Lu]Lu-PSMA-based RLT compared to transitioning to androgen receptor targeted therapy (ARTT).A recent press release from the SPLASH study demonstrated improved radiographic progression-free survival in mCRPC patients who progressed on ARTT and were then treated with RLT rather than switching to ARTT.While these investigations mostly focus on beta emitters, a corresponding concept for alpha emitters is emerging, while research in this field is still in its early stages.In a study evaluating the tandem utilization of [ 177 Lu]Lu-PSMA-617 and [ 225 Ac]Ac-PSMA-617 in patients with early-stage hormone-sensitive metastatic prostate cancer, 85% of patients exhibited a PSA response of ≥50% following PSMA-RLT [80].This raises hope that future studies evaluating 225 Ac based RLT alone or in combination with beta emitters might be applied in early prostate cancer.

Current and Future Combination Therapy Approaches in PCa Treatment
Research in the field of targeted radioligand therapy led to clinical approval of agents such as [ 177 Lu]Lu-DOTATATE, 131 I-metaiodobenzylguanidine, Bexxar, and Zevalin, for treating conditions like neuroendocrine tumors (NETs), neuroblastoma, and non-Hodgkin lymphoma.Monotherapy, though the cornerstone, has been challenged by the adaptive plasticity exhibited by prostate cancer cells [81].These cells, adept at reconfiguring their signaling networks, often render single-agent interventions ineffective over time.Given the limitations of monotherapy, various combination therapy strategies are being explored, including enhancing tumor perfusion to enable optimal radiopharmaceutical distribution, upregulating target receptors to augment cellular uptake, combining nuclide therapy with DNAdamaging drugs, inhibiting essential processes like DNA damage repair for radiosensitization, and incorporating immune checkpoint inhibitors (Figure 6).One of the approaches is to combine mAb and the small molecule with two different therapeutic radioisotopes targeting the same protein on the cell surface.In this context, the use of mAb ([ 225 Ac]Ac-J591) and [ 177 Lu]Lu-PSMA-I&T showed improved binding and higher radiation dose delivery compared to a single agent, resulting in a synergistic therapeutic efficiency in xenograft models.Clinical studies are in progress on mCRPC patients to find the maximum tolerated dose for the combination treatment of PSMA targeting [ 225 Ac]Ac-J591 and [ 177 Lu]Lu-PSMA-I&T (NCT04886986) [70,72].As TAT is known to produce DNA damage as well as activation of immunogenic response, the use of [ 225 Ac]Ac-J591 in combination with pembrolizumab (PD-1 inhibitor) is being tested in PCa patients (NCT04946370) [76].
Since the poly (ADP-ribose) polymerase (PARP) enzymes are the first responder to DNA damage, alpha particle therapy may be effectively combined with the PARP inhibitors (PARPi) (Figure 6). 225Ac, an alpha particle-emitting radionuclide, contributes to this process by inducing highly localized, dense ionization along its path, resulting in complex DNA double-strand breaks.PARPi disrupt DNA repair pathways, particularly in cancers with BRCA mutations, leading to the accumulation of lethal DNA damage.By concurrently inhibiting PARP and exposing cancer cells to the high LET radiation from 225 Ac, a dual assault on DNA repair mechanisms is orchestrated.The combination of beta particle therapies, with PARPi, like Olaparib, has been a subject of research interest [82,83].A recent phase I study specifically investigated the safety profile of combining Olaparib with 223 Ra in patients with mCRPC, indicating the growing interest and exploration of this combination in clinical settings [84].

Actinium-225 production
The main source of 225 Ac has been the stocks of 229 Th extracted from 233 U.The milking of 225 Ac derived from 229 Th remains insufficient to facilitate the widespread and routine demand.Due to this, alternative approaches to produce 225 Ac at a large scale are being explored.The accelerator produced 225 Ac has potential to meet current demand.Firstly, the high-energy proton irradiation (>100 MeV) of 232 Th produces a large quantity of 225 Ac, but it concurrently yields other radionuclides through spallation and fission reactions that need to be separated [18,85].Notably, a longer-lived 227 Ac isotope, with a half-life of 21.8 years, co-produced at a rate of 0.1-0.2%relative to 225 Ac activity, is the main limitation.In addition to this, efforts are being made to produce 225 Ac through proton irradiation of 226 Ra targets.The most promising approach is the use of medium energy proton (20 MeV) accelerators.The irradiation of 226 Ra targets via the reaction 226 Ra(p,2n) 225 Ac presents distinct merits compared to the 232 Th spallation process [86].It can be performed in medium-sized cyclotrons, making it cost-effective without compromising the yield.Proton irradiation of 226 Ra targets yields pure 225 Ac without co-production of 227 Ac.Although short-lived impurities like 226 Ac (with a half-life of 29 hours) and 224 Ac (with a half-life of 2.9 hours) are formed, their presence is manageable.Another approach to produce the 225 Ac is via photonuclear 226 Ra(ɣ,n) 225 Ra reaction, where generated 225 Ra decays to 225 Ac [87].This approach will produce the 225 Ac without impurities, however in lower yield [87].
In response to the growing worldwide demand and the critical scarcity of this isotope, the United States Department of Energy's Isotope Program (DOE IP) has initiated a comprehensive array of initiatives.These initiatives include strategic facility utilization and judicious allocation of funding, spanning various avenues for the synthesis of 225 Ac [88].Of particular significance among these ventures is the "Ac-225 Tri-Lab Effort," a synergistic collaboration undertaken by three national laboratories: Oak Ridge National Laboratory (ORNL), Brookhaven National Laboratory (BNL), and Los Alamos National Laboratory (LANL).This collaborative endeavor delves into the feasibility of generating 225 Ac via accelerator-based methodologies, thereby contributing substantively to addressing the augmented global need.TRIUMF, a Canadian physics laboratory, has also been working towards production of 225 Ac and other isotopes.Due to the high costs associated with each batch of 225 Ac production, the current approach for synthesizing 225 Ac at TRIUMF involves in-house production using the Isotope Separator and Accelerator (ISAC) Facility [19].At TRIUMF, the 225 Ac is generated through the irradiation of Uranium and Thorium targets with protons, and after additional purification steps, it is employed in TAT studies.

Quality control of 225 Ac radiopharmaceuticals
The radiolabeling reactions and percentage purity of 225 Ac compounds are primarily monitored using radio-thin layer chromatography (radio-TLC).The continuous generation of daughter isotopes ( 213 Bi and 221 Fr) makes it challenging to accurately estimate the radiochemical yield immediately after the run. 221Fr, with half-life of 4.9 minutes, reaches secular equilibrium in approximately 1 hr, while 213 Bi (t1/2=45.6minutes) take hours.Therefore, it is advisable to read the radio-TLCs after attaining secular equilibrium, approximately 4 to 5 hours after the completion of radio-TLC [89].International Atomic Energy Agency (IAEA) also recommends radio-TLC as a quality control test for 225 Ac labeled peptides [90,91].A study by Yang et al. reported that additional quality control tests are needed to use of any radiopharmaceutical with 225 Ac [91].The [ 225 Ac]Ac-crown-αMSH molecule synthesized by Yang et al showed radiolysis due to the high-energy alpha particles, which was not observed on radio-TLC but was evident on radio-high performance liquid chromatography (radio-HPLC).Therefore, the combination of delayed iTLC for radiolabeling, HPLC for the integrity of radiopharmaceuticals, and gamma spectroscopy for the determination of free 225 Ac are recommended [91].Additionally, retention times of the 213 Bi and 221 Fr, determined from fraction collection and gamma counting, could be used to calculate the radiochemical purity using HPLC [91].Furthermore, the use of ROS scavengers like ascorbic acid or reducing the time between purification and dose injection could help reduce radiolysis [91,92].Since the daughters 213 Bi and 221 Fr take different times to reach secular equilibrium with 225 Ac, quantification of 225 Ac using 213 Bi and 221 Fr before equilibrium or in case of 213 Bi redistribution is challenging.In this regard, to determine the 225 Ac amount, Seone et al. developed a multi-time point gamma counting of 213 Bi (before secular equilibrium), and single time-point gamma counting of 213 Bi and 221 Fr considering no secular equilibrium [93].Along with this, one major quality control concern related to accelerator produced 225 Ac via spallation of 232 Th is an impurity of long lived 227 Ac.Various purification methods are being tested to separate the 227 Ac from 225 Ac [18,85,94].

Recoiling energy, toxicity, and side effects
The toxicity associated with 225 Ac-based TAT may arise in part from the recoil effect.Although significant progress has been made in developing chelators that create stable and inert complexes with 225 Ac, the challenge of the recoil effect remains [20].This recoil effect results in the release of the 221 Fr isotope from the chelate as a result of the alpha emission from 225 Ac, abiding by the principles of momentum conservation.During the decay process to produce four net alpha particles, the energy released due to the recoil of daughter nuclides exceeds that of more than 10,000 chemical bonds, thereby leading to the separation of daughter nuclides from the chelator (Figure 7A) [21].This leads to the distribution of daughter isotopes to non-targeted tissues causing significant toxicity to healthy organs and compromising therapeutic efficacy.Various preclinical and clinical studies report that redistribution of daughter 213 Bi into the kidney from 225 Ac radioligand molecules can be observed, in some cases causing nephrotoxicity (Figure 7B) [36,50,95,96].
To address the toxicity associated with recoiled daughters, researchers are investigating novel strategies (Figure 8).One approach involves facilitating the rapid uptake and internalization of alpha emitters within the target tissue (Figure 8A) [97].The use of high affinity and rapidly internalizing molecules may decrease the delivery of daughter isotopes to healthy organs [21,97,98].Secondly, the encapsulation of the nuclide within nanoparticles could minimize damage to normal cells (Figure 8B).Preclinical studies have explored the feasibility of loading radioisotopes in nanoformulations.Nanozeolites were successfully used as a carrier for 224 Ra and 225 Ra, along with their daughter radionuclides [99].Similarly, surface-modified, PSMA-targeted liposomes also demonstrated the usability of nanoformulations for the delivery of 225 Ac to prostate cancer cells [100].In another approach, the layered nanoparticles consisting of 225 Ac-doped {La0.5Gd0.5}PO4@GdPO4@Auwere prepared to trap the daughters inside the core after decay [101].These gold-coated, layered nanoparticles, labeled with 225 Ac, help retain 213 Bi in the target tissue minimizing the kidneys uptake over time compared to the non-coated or non-targeted nanoparticles [101].Nano formulations allow site-specific and controlled delivery of the payload to target site.Thus, liposomes were utilized to deliver 225 Ac and its radioactive daughters to ovarian cancer cells.The daughter isotopes were successfully retained in the liposome core, however the encapsulation efficiency was low [102].Similarly, folate-F(ab′)2 decorated liposomes allow targeted 223 Ra delivery [103].In addition to targeting efficiency, the subcellular distribution of therapeutic molecules is crucial.A comparison was conducted to evaluate the cytotoxicity of PSMA and PSMA antibody-decorated liposomes loaded with 225 Ac [104].The PSMA-decorated liposomes were three times more effective in inducing cell death, attributed to the delivery of the isotope to the perinuclear space.Ensuring stringent quality control measures is imperative in the development of nanocarriers for 225 Ac delivery.Stability studies under relevant physiological conditions are essential to ascertain the long-term viability and performance of the nanocarriers.Addressing quality control and stability issues is paramount to guarantee the safety and efficacy of 225 Ac-loaded nanocarriers, which could pave the way for their future translation into clinical applications [104][105][106].Extensive research on radionuclide carriers, primarily antibodies, has predominantly focused on IgG-type antibodies.However, the commercial viability of radiolabeled antibodies faces challenges due to hematological and other toxicities associated with the prolonged circulation of antibodies [107].To overcome these challenges, alternative strategies including antibody fragments, recombinant proteins, and pre-targeting approaches have been explored.A noteworthy strategy is a pretargeting, where tumor cells are initially targeted with an unlabeled antibody [108].Subsequent administration of a radiolabeled compound binds to the antibody, facilitating targeted delivery (Figure 8C).Unbound radiolabeled molecules are swiftly excreted, contributing to a more precise and efficient therapeutic approach [109].Hapuarachchige et.al studied a pretargeting approach on PC3 cells where an anti-PSMA 5D3 mAb and its F(ab')2 fragments with trans-cyclooctene (TCO) were used as a pretargeting component [110].Subsequently, a drug delivery component, human serum albumin loaded with the DM1 anti-tubulin agent functionalized with PEGylated tetrazine (PEG4-Tz), was employed.The results demonstrated enhanced specific killing of PSMA(+) cells.Similarly, van Rij et al. employed the trivalent bispecific antibody TF12, composed of two anti-TROP-2 Fab fragments and one anti-histamine-succinyl-glycine (HSG) Fab fragment, for pretargeted theranostics.The delivery of the therapeutic agent, a radiolabeled hapten peptide ([ 177 Lu]Lu-IMP288), demonstrated efficacy in treating TROP-2-expressing PC3 tumors [111].PET imaging using the same pair, [ 68 Ga]Ga-IMP288 and TF12, enabled the detection of PCa tumors with improved contrast, showing lower bladder or kidney uptake compared to the commonly observed pattern with [ 68 Ga]Ga-PSMA or FDG [111].Similar to this, a theranostic pair, using proteus-DOTA as a carrier for 225 Ac and 111 In was developed [112].This carrier was captured in vivo by a single-chain variable fragment (scFv) attached to the cell surface.This pretargeting study revealed high tumor accumulation and low uptake in normal tissues.The results were reproduced in solid human cancer xenograft models, including colorectal cancer, breast cancer, and neuroblastoma.
By injecting a radiolabeled molecule locally into the tumor tissue, redistribution of daughter isotopes can be minimized to some extent (Figure 8D) [21].The dose escalation study by Krolicki et.al for [ 225 Ac]Ac-DOTA-SP have shown that the local injections of a radiopharmaceutical were safe for glioblastoma patient when administered at 10, 20 and 30 MBq per cycle for 6 cycles (2 month interval) [113].No hematological or renal toxicities were reported from this study [113].Similar to this, in a pilot study by Cordier et al. in five patients of gliomas were locally injected with [ 213 Bi]Bi-DOTA-substance P. The [ 213 Bi]Bi-DOTA-substance P treatment was found to be well-tolerated without toxicity [114].While direct administration is suitable for short half-life isotopes, caution is crucial for long half-life isotopes.The direct local administration route has been employed in the treatment of lung and pancreatic cancers in vivo [115,116].The prolonged presence of long-lived isotopes in the body increases the risk of cumulative radiation exposure to healthy tissues.Therefore, meticulous consideration and potentially modified administration strategies are essential to ensure the balance between therapeutic efficacy and minimizing adverse effects when dealing with long half-life isotopes.

Radiobiological considerations, dose optimization, and fractionation
As the evidence supporting the utilization of 225 Ac and other high-LET radioisotopes in cancer therapy accumulates, the consideration of the relative biological effectiveness (RBE) value becomes imperative [117].This significance is particularly highlighted when patients undergo one or more forms of radiation therapy, such as a combination of EBRT followed by TAT, or when patients resistant to [ 177 Lu]Lu-PSMA-617 treatment are enrolled for [ 225 Ac]Ac-PSMA-617 therapy.The high RBE values characteristic of high-LET isotopes stem from their induction of DNA double-strand breaks (DSBs), ultimately leading to cell death.Despite technological advancements allowing for escalated tumor doses, the current approach often involves prescribing the same dose uniformly to all patients, this could lead to underdosage or overdosage for specific patients.Consideration of the distribution and volume of metastases can help tailor TAT treatments to individual patients, optimizing efficacy and minimizing toxicity.Thus, studying the tumor cells before and after the radiation dose would help prescribe an individual radiation dosage.Moreover, the bystander effects of alpha therapy, which can trigger immune responses and non-targeted tumor-cell killing, may offer promising therapeutic benefits.
In the context of therapeutic dose vs toxicity, fractionation of a large therapeutic dose of 225 Ac into smaller manageable doses could enhance safety.This fractionation strategy serves several purposes.Firstly, it helps reduce the potential for toxicity by allowing healthy tissues to recover between doses.Secondly, it provides a more precise and controlled approach to treatment, optimizing the therapeutic ratio, and maximizing the impact on cancer cells while minimizing harm to surrounding normal tissues.Thirdly, it aligns with radiobiological principles, taking advantage of the fact that cancer cells may be more susceptible to cumulative radiation damage over multiple exposures.The fractionated dose strategy for 225 Ac has been utilized in prostate cancer and multiple myeloma with promising results [36,70,118,119].While it is widely acknowledged that fractionation of therapy offers advantages, it is important to consider potential disadvantages as well.Fractionation, although beneficial, can present challenges such as complex implementation strategies, treatment interruption, increased cost, and the potential for delayed tumor regression.

Ac Imaging techniques
The four alpha particles from the decay chain of 225 Ac imparts a high therapeutic efficacy; however, current studies indicate the hurdles of targeting affected tissue with the entire decay chain.Thus, it becomes imperative to confirm the presence of the daughter isotopes at the target tissue after the injections of 225 Ac-based radiopharmaceuticals.In preclinical studies, the ex-vivo biodistribution analysis of 213 Bi and 221 Fr is difficult to complete due to their short half-lives.The gamma emissions of daughters, including the 440 keV ( 213 Bi) and 218 keV ( 221 Fr) peaks for SPECT imaging, remain valuable for dosimetry assessment and therapy response evaluation; however, the low therapeutic 225 Ac activity administered, typically 100 kBq/kg, result in limited gamma emissions for imaging [120].Alternatively, much progress has been made in the small-scale organ dosimetry for tumor models used in preclinical studies, which can be extrapolated to human subjects [121].This involves ex-vivo autoradiography of alpha particle decay events, generating a real-time dose-rate map that provides information about the spatial distribution of the radioactivity in the tissue [121].Traditional collimator-based SPECT imaging has challenges to image gamma emission above 300 keV, therefore utilization of 440 keV from 213 Bi is more difficult.To overcome this, a new approach to combine the coded aperture imager (for lower keV) and Compton imager (for higher keV) was utilized to visualize and quantify the daughters from 225 Ac [122].
Quantitative single photon emission computed tomography (SPECT) of gamma emissions at 440 keV and 218 keV peaks allows image-based dosimetry to determine the absorbed dose.Although posttherapy imaging is challenging for alpha emitters, one study presented a case of mCRPC treated with [ 225 Ac]Ac-PSMA-617.The posttherapy scans, utilizing 3 different energies 78, 218, and 440 keV showed successful targeted therapy, and distribution was visualized [123].Imaging with 3 major photopeaks (78, 218, and 440 keV) enhanced image quality compared to the previously reported 440 and 218 keV photopeaks [124].Recently, a quantitative SPECT imaging for [ 225 Ac]Ac-PSMA-I&T therapy for mCRPC patients was demonstrated with image-based estimations of the absorbed dose [125].These prior and ongoing studies indicate promise for direct imaging of the daughters of 225 Ac.

Thorium-227
Targeted Thorium-227 conjugates (TTCs) represent another class of therapeutic radiopharmaceuticals meticulously designed for precision in TAT.The TTC process involves binding the alpha-emitting Thorium-227 ( 227 Th) to a chelator, strategically linked to a monoclonal antibody (MAb) or small molecules engineered for tumor targeting [126]. 227Th, belonging to the actinide series (half-life of 18.7 days), follows a decay scheme featuring a cascade of alpha (five alpha particles) and beta emissions, culminating in the formation of the stable isotope 207 Pb (Figure 9) [127].The alpha particle emitted during the decay of 227 Th possesses a mean energy of 5.9 MeV, contributing to a total energy of 34 MeV from all the five alpha particles in the decay series.Additionally, 227 Th serves as the precursor to Radium-223 (Xofigo), an alpha-emitting radionuclide globally approved for clinical use in CRPC.In contrast to 223 Ra, the precursor radionuclide 227 Th demonstrates the ability to form stable chelator complexes, making it well-suited for targeted radioimmunotherapy.The extended half-life of 227 Th not only streamlines practical handling and administration but also proves advantageous for achieving elevated radiation doses at the tumor site.This prolonged half-life contributes to enhanced efficiency in the production process and allows for optimized scheduling in therapeutic applications.
The radiolabeling of 227 Th has posed a substantial hurdle in the progress of TTC.Initial investigations utilizing DOTA necessitated elevated temperatures (60 o C) for efficient radiolabeling, a condition poorly suited for macromolecules like antibodies [128,129].This underscores the demand for innovative methodologies to improve radiolabeling efficiency, particularly when dealing with larger biological entities.To overcome this challenge, Ramdahl et al. introduced a solution by employing HOPO (2,3-hydroxypyridinone) for the chelation of 227 Th at room temperature, offering a promising avenue for addressing the issues in the context of TTC development [130].Recently, Hammer et al. reported the use of PSMA-TTC for the treatment of preclinical models of PCa [131].PSMA-TTC was synthesized by conjugating the HOPO chelator to PSMA targeting full-length antibody (BAY 2315158) for radiolabeling.This PSMA-TTC exhibited selective binding and internalization in the cells expressing PSMA, resulting in DNA damage and apoptosis from alpha particle-mediated toxicity.PSMA TTC was also effective for the treatment of AR-sensitive (LNCaP and ST1273) and AR-resistant (KUCaP-1) in-vivo tumor models.The conjugate was specifically effective in inhibiting cancer progression and abnormal bone growth in a bone metastasis model [131].
Given that PCa predominantly depends on androgens for growth, concurrently addressing androgen receptor signaling alongside PSMA-TTC treatment emerges as a promising approach for prostate cancer therapy.Hammer et al. expanded their investigations to assess the synergies between PSMA-TTC and darolutamide, an androgen receptor inhibitor [132].The combined therapy involving PSMA-TTC and darolutamide exhibited synergistic effectiveness in the in-vitro as well as in-vivo tumor models.AR inhibition from darolutamide increased the PSMA expression thereby increasing the tumor-targeted delivery of PSMA-TTC and the therapeutic response [132].Currently, the clinical trial to test the safety, tolerability, pharmacokinetics, and antitumor action of a PSMA-TTC is ongoing (NCT03724747).In addition to this, a combination of the PARP inhibitor (Olaparib) and PSMA-TTC is also being tested with synergistic anti-tumor results in various tumor models [133].In addition to this, development of novel peptidomimetic small molecules is also being designed to target PSMA for the delivery of 227 Th.A recent study by Böhnke et al. involved the synthesis of mono-and multimeric (di-, tri-, and tetramers) PSMA-targeting small molecules with modified carboxylated HOPO for the treatment of PCa [134].The synthesized carboxy-HOPO exhibits heightened hydrophobicity and demonstrates robust labeling efficacy at room temperature.Among the studied forms of PSMA TTC, both monomeric and dimeric configurations exhibited exceptional tumorto-background ratios with minimal accumulation in the kidneys.In contrast, the larger size of the trimer and tetramer led to increased accumulation in the liver [134].Despite challenges in radiolabeling, innovative approaches like HOPO chelation at room temperature and synergistic treatments with androgen receptor inhibitors offer exciting avenues for further development and clinical exploration.

Radium-223
The ALSYMPCA trial played a pivotal role in establishing the therapeutic merit of Radium-223 (Xofigo), leading to its regulatory approval and emergence as a groundbreaking intervention for individuals with bone metastases.Radium-223's remarkable ability to selectively target bone metastases has led to improved overall survival rates and a significant delay in the development of symptomatic skeletal lesions [135].As illustrated in Figure 9 decay scheme, 227 Th undergoes alpha particle emission to transform into 223 Ra, initiating a shared decay pathway between 227 Th and 223 Ra with the remaining daughter isotopes. 223Ra has a half-life of 11.4 days, resulting in a net of four alpha particles and two beta particles.Injection of the 223 Ra and 89 Sr resulted in increased bone uptake in the osseous site, without affecting the normal bone marrow suggesting its bone-seeking property [136].Subsequent preclinical investigations consistently validated the bone-targeting efficacy of 223 Ra chloride in metastatic tumors [137].The clinical trials focused on toxicity and dose estimations and therapeutic effects of administration of Radium-223 to prostate cancer patients proved the efficacy for use in humans [138][139][140].The unique bone-targeting properties of 223 Ra, demonstrated through preclinical investigations and clinical trials, indicate its efficacy and safety in treating prostate cancer patients.

Astiatine-211
Astatine-211 ( 211 At) stands out as a compelling TAT radioisotope, presenting a judicious balance of favorable characteristics [141].With a half-life of 7.2 hours, 211 At is endowed with an optimal temporal window for therapeutic applications.Importantly, unlike the other TAT isotopes, its decay series exhibit an absence of toxic daughters, a crucial consideration in ensuring the safety and efficacy of cancer treatments.Furthermore, 211 At offers the practical advantage of feasible production in clinically relevant amounts, enhancing its accessibility for potential clinical applications.These attributes collectively position 211 At as a promising contender in the expanding range of alpha-emitting radionuclides for targeted therapies.The decay process of 211 At involves two branches (Figure 10).The first decay branch (42% decay) leads to the generation of 207 Bi (t½ = 32.9years) and the emission of an α-particle (5.9 MeV).The 207 Bi subsequently decays by electron capture to 207 Pb.The second decay branch (58% decay) facilitated by electron capture produces 211 Po (t½ = 516 milliseconds), which subsequently undergoes decay to 207 Pb, emitting an α-particle (7.4 MeV).  21At by electrophilic astatodestannylation [143].These urea-based molecules showed tumor accumulation with various degrees of dehalogenation resulting in thyroid, kidney, and stomach accumulation [142,143].The radiolabeling method utilized in these studies results in lower molar activity.To address this challenge, a new methodology has been presented by Shirakami et al. for the synthesis of PSMA-targeted ligands [144].This innovative approach entails a substitution reaction wherein dihydroxyboryl groups are replaced with 211 At on PSMA ligands.Although the synthesized molecules ([ 211 At]At-PSMA1, [ 211 At]At-PSMA5, and [ 211 At]At-PSMA6) show high tumor retention; due to dehalogenation, as well as due to PSMA expression at proximal tubules, significant toxicity was seen in healthy organs including kidneys [145,146].In addition to this, peptide formulation labeled with the 211 At has been tested to achieve TAT by targeting bombesin receptor [147].The bombesin targeting [ 211 At]At-AB-3 molecules failed to accumulate in the tumor to achieve sufficient concentration, possibly due to stability and dehalogenation issues.
To address potential PSMA negativity and heterogeneity, Back et al. tested the anti-PSCA A11 minibody to achieve targeted delivery of the 211 At to prostate stem cell antigen (PSCA) [148].PSCA is overexpressed in localized as well as metastatic prostate cancer.[ 211 At]At-A11 showed significant therapeutic action for both subcutaneous as well as tibia tumor models.Interestingly, the blocking of sodium iodine importer with sodium perchlorate significantly reduced the uptake of free 211 At in healthy organs, minimizing the off-target toxicity.In summary, 211 At shows promise for Targeted TAT in PCa with favorable features, optimal timing, and no toxic byproducts.

Bismuth-213
The integration of Bismuth-213 ( 213 Bi, half-life= 45.6 minutes) into TAT gained significant traction through the pioneering efforts of the Memorial Sloan Kettering Cancer Center in developing a 225 Ac/ 213 Bi generator.This generator, a pivotal advancement, enabled on-site production of 213 Bi, thereby improving accessibility for its application in the treatment of patients with leukemia [149,150].Early investigations using 213 Bi-labeled J591 ([ 213 Bi]Bi-J591) demonstrated encouraging outcomes in terms of targeted binding and effective tumor cell eradication in in-vitro cells, spheroids, and in-vivo tumor models [151][152][153].These findings affirmed the viability of 213 Bi in TAT, particularly for metastatic microtumors, where high doses of radiation per cell are crucial for achieving complete eradication of tumor cells, additionally, highlighting that larger solid tumors show response but the complete regression may not be achieved [154,155].In addition to PSMA targeting with J591, another potential target investigated for the delivery of 213 Bi is the plasminogen activator inhibitor type 2 (PAI2) protein, known to be overexpressed on prostate cancer cells.The [ 213 Bi]Bi-PAI2 conjugate demonstrated induced cell death through mechanisms involving DNA damage and apoptosis, while concurrently exhibiting low or negligible toxicity [154].Yong Li and colleagues proposed the concept of multiple targeted radioimmunotherapy conjugates, wherein multiple proteins were targeted using 213 Bi radiolabeled vectors for respective proteins [155][156][157].The cocktails of 213 Bi-labeled monoclonal antibodies against C595, BLCA-38, and J591, along with [ 213 Bi]Bi-PAI2, were tested on PCa cells, suggesting that the cocktails exhibited superior efficacy compared to monotherapies.However, for the personalized application of these combinations, information on target expression is imperative to determine the combination that would work effectively [155,156,158].
In addition to the high molecular weight macromolecules, utilizing small molecular weight vectors like PSMA or nanobodies for 213 Bi delivery offers distinct advantages [159].These vectors, due to their compact size, enhance tissue penetration, exhibit rapid clearance from non-target sites, and efficiently distribute within tumors.Additionally, the biological half-life of the vector aligns with that of 213 Bi, resulting in rapid blood clearance reducing radiation exposure, and lowering the risk of hematologic toxicity [160].In this context, the first-in-human study with [ 213 Bi]Bi-PSMA-617 showed a remarkable response (PSA decreased to 43 µg/mL from 237 µg/L) when treated with two cycles of a combined 592 MBq dose [161,162].Additionally, patients resistant to [ 177 Lu]Lu-PSMA-617 exhibited a substantial anti-tumor response with minimal hematological toxicity when treated with [ 213 Bi]Bi-DOTATOC [163].An additional advantage of 213 Bi, in comparison to 225 Ac, is its absence of daughter isotopes, eliminating concerns related to recoiling.This characteristic simplifies the dosimetry calculations and ensures a more controlled and predictable radiation delivery in targeted alpha particle therapy.A key challenge in advancing 213 Bi-based TAT lies in the availability of a reliable 225 Ac/ 213 Bi generator.
The successful establishment and operation of a reliable 225 Ac/ 213 Bi generator are indispensable for securing a sustainable and accessible supply of 213 Bi, thereby facilitating the ongoing advancement and widespread adoption of this promising therapeutic modality [18,164].The growing number of sites dedicated to accelerator-produced 225 Ac holds promise in addressing and resolving this challenge, offering a potential solution to ensure the availability and accessibility of 213 Bi for medical applications.

Lead-212
An additional noteworthy in situ α-emitting radiometal is Lead-212 ( 212 Pb, 10.6 days half-life).Its decay involves α and β− particle emission to produce stable 208 Pb.Utilizing 212 Pb offers the advantage of employing the 203 Pb as an imaging surrogate, facilitating imaging and dosimetry estimations.In clinical settings, 212 Pb labeled trastuzumab has been tested in ovarian cancer patients with peritoneal carcinomatosis, and it demonstrated notable antitumor activity with good tolerability [165,166].Similarly, in a preclinical study, a single intravenous dose of [ 212 Pb]Pb-trastuzumab resulted in a substantial reduction in tumor growth with prolonged survival in mice with PC3 tumors, importantly, without loss of body weight [167].The first human study with PSMA targeted 212 Pb involved the use of urea-based ligands with p-SCN-Bn-TCMC or DO3AM chelators [168].Two patients with mCRPC were injected with 203 Pb labeled CA012, and dosimetry estimates were acquired, revealing an estimated effective dose of 6-7 mSv from 250-300 MBq of [ 203 Pb]Pb-CA012.In-vivo studies have also explored the use of 212 Pb for labeling PSMA-directed small molecules or biomarker-specific antibodies for the treatment of preclinical prostate cancer tumors [159].

Conclusion
Recent and notable clinical outcomes in the treatment of advanced-stage cancers with beta-and alpha-emitting radiopharmaceuticals have sparked increased interest in radiopharmaceutical therapy.The approval of [ 177 Lu]Lu-PSMA-617, coupled with emerging evidence in receptor-targeted therapy that capitalizes on the unique properties of cancer cells, underscores the potential for developing novel therapeutic strategies.Actinium-225, known for high LET, longer half-life, and shorter path length has emerged as an important alpha emitter for various malignancies, including prostate cancer.Therefore, [ 225 Ac]Ac-PSMA-617 therapy results have been considered as one of the promising options for treatment-or [ 177 Lu]Lu-PSMA-617-resistant patients.To enhance the therapeutic efficacy of PSMA-based radionuclide therapy, diverse strategies are being explored in clinical settings.These strategies encompass developing refined patient selection methods, escalated radiation damage through dosimetry-guided dose selection, or utilization of α-emitters in place of β-emitters.Additionally, combined approaches aimed at overcoming radioresistance mechanisms, whether intrinsic or acquired, are being investigated.These multidimensional strategies involve novel hormonal agents, PARP inhibitors, and immunotherapy, all geared toward enhancing the effectiveness of PSMA-based radionuclide therapy.However, challenges persist as a vast number of mCRPC patients show no response to [ 177 Lu]Lu-PSMA-617 RLT.Heterogeneity in PSMA expression, mechanisms of resistance, and the potential impact of PSMA-targeted therapies on non-prostate tissues necessitate further investigation.While [ 177 Lu]Lu-PSMA-617 and [ 225 Ac]Ac-PSMA-617 treatments have enhanced the overall prognosis for patients, the emergence of resistance within tumors has remained an issue, leading to ongoing disease progression.The length of responses to RLT is frequently brief, even among patients who are initially responsive, and the underlying reasons for resistance remain unknown.Inadequate radiation dose delivery has been established as a factor contributing to resistance, particularly for RLT with β-emitters.Diffuse marrow infiltration is a typical post-treatment progression pattern with [ 177 Lu]Lu-PSMA-617, which could be caused by insufficient radiation dose distribution to small-volume illness.Given that [ 177 Lu]Lu-PSMA-617 treatment failure is frequently related to the advancement of micrometastatic illness, the use of other radionuclides with shorter route lengths may be a viable approach.Single traversals of alpha particles or Auger electrons can trigger cytotoxic double-stranded DNA breaks in the nucleus, thereby bypassing the constraints associated with micrometastatic disease resistance shown with [ 177 Lu]Lu-PSMA-617.
The successful development of radiopharmaceuticals relies on efficient chelators and tumor-specific targeting vectors.Chelators tested for radiolabeling of 225 Ac are EDTA, PEPA, TETPA, DTPA, DOTA, and Macropa, among others.Among these, the DOTA and Macropa are the most favored to produce higher radiolabeling yield with suitable stability.The review brought attention to the significance of biomarkers in prostate cancer, with a particular focus on the commonly used biomarker PSMA.Despite its conventional use, ongoing studies to develop alternative PSMA ligands, peptides, or albumin modifications underscore the continuous exploration and refinement of diagnostic approaches related to PSMA.As research advances, the identification and utilization of biomarkers will likely play a pivotal role in enhancing diagnostic accuracy and tailoring more effective therapeutic interventions.In this regard, various new biomarkers are being tested for PSMA-negative prostate cancer, most notable novel biomarkers being tested are CD46, human KLK3, and VE-cadherin.DLL3, CDCP1 or GRPR are a few other promising targets that can be used for alpha therapy in PSMA-negative tumors.
The review highlights challenges in 225 Ac-based therapies, including isotope production, recoiling energy, daughter redistribution, and the need for improved imaging techniques.These hurdles represent crucial areas for further research and development to unlock the full potential of 225 Ac-based therapies.To improve the efficacy of current TAT agents, various combination approaches were discussed including combinations with PARP inhibitors, anti-androgens, and immune modulators.We also shed light on alternative isotopes, such as 227 Th, 223 Ra, 211 At, 213 Bi, 212 Pb, and 149 Tb suggesting a diversified landscape for potential alpha-emitting radionuclides in TAT for prostate cancer.These alternatives provide avenues for further research, offering potential solutions to some of the obstacles associated with 225 Ac.While the application of actinium-225 in prostate cancer therapy holds tremendous potential, further research and development are vital to address the current limitations and optimize its use.The evolving landscape of alpha-particle therapy, with 225 Ac at its forefront, signifies a paradigm shift in the way we approach and treat prostate cancer.As research continues to advance, the prospect of realizing the full therapeutic potential of actinium-225 in clinical settings becomes an exciting and achievable goal.

Figure 1 :
Figure 1: Schematic illustration of radioligand therapy molecules: A. Binding of small molecule PSMA ligands to receptor active site on the cell surface.B. Representation of the use of peptide-based targeting agents.C. Tumor-specific binding of antibodies to the extracellular domain of surface proteins.

Figure 2 :
Figure 2: Schematic comparison of the distance traveled and Linear Energy Transfers (LETs) of α, β particles, and Auger electrons in tumor and healthy tissues.

Figure 3 :
Figure 3: Decay scheme of 225 Ac showing daughter isotopes, alpha, and beta particle emissions along with the energies.

Figure 4 :
Figure 4: Chronological sequence of significant milestones spanning from the initial identification of 225 Ac to its current role in prostate cancer.

Figure 5 :
Figure 5: Chemical structures of the chelators used for 225 Ac radiolabeling.

Figure 6 :
Figure 6: Summary of possible combination therapy approaches to enhance the therapeutic effect of TAT.

Figure 7 :
Figure 7: The decay process of 225 Ac, leading to the emission of alpha particles, induces recoiling energy, subsequently causing the redistribution of daughter isotopes.A. Upon injection of the 225 Ac-based TAT radiopharmaceutical, accumulation occurs in the tumor tissue.The 225 Ac in circulation or tumor tissues decays, producing alpha particles along with recoiled 221 Fr and 213 Bi.B. The redistribution of the 213 Bi contributes to nephrotoxicity.

Figure 8 :
Figure 8: Strategies to mitigate the toxicity of recoiled daughters.A. Use of rapid uptake and internalizing targets, B. Encapsulation of the TAT radionuclide within nanoparticles, C. pretargeting, D. Intra-tumoral administration of radioactivity via injection.

Figure 9 :
Figure 9: Schematic presentation of the decay series of Thorium-227 and Radium-223.
. Following this, McDevitt et al.

Table 1 :
Radiolabeling conditions and radiochemical yields for chelators used for 225 Ac complexes.